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Background: Astaxanthin is a red pigment required by feed, nutraceutical, and cosmetic industries for its pigmentation and antioxidant properties. This carotenoid is one of the main high-value products that can nowadays be derived from microalgae cultivation, raising important industrial interest. However, state-of-the-art astaxanthin production is the cultivation of the green alga Haematococcus pluvialis (or lacustris), which faces high costs and low production yield. Hence, alternative and efficient sources for astaxanthin need to be developed, and novel biotechnological solutions must be found. The recently discovered cyanobacterium, Synechococcus sp. PCC 11901 is a promising photosynthetic platform for the large-scale production of high-value products, but its potential has yet to be thoroughly tested. Results: In this study, the cyanobacterium Synechococcus sp. PCC 11901 was engineered for the first time to our knowledge to produce astaxanthin, a high-value ketocarotenoid, by expressing recombinant β-ketolase (bKT) and a β-hydroxylase enzymes (CtrZ). During photoautotrophic growth, the bKT-CtrZ transformed strain (called BC) accumulated astaxanthin to above 80% of the total carotenoid. Moreover, BC cells grew faster than wild-type (WT) cells in high light and continuous bubbling with CO2-enriched air. The engineered strain reached stationary phase after only 4 days of growth in an airlift 80-mL photobioreactor, producing 7 g/L of dry biomass, and accumulated ~ 10 mg/L/day of astaxanthin, which is more than other CO2-consuming multi-engineered systems. In addition, BC cells were cultivated in a 330-L photobioreactor to link lab-scale experiments to the industrial scale-up. Conclusions: The astaxanthin volumetric productivity achieved, 10 mg/L/day, exceeds that previously reported for Haematococcus pluvialis, the standard microalgal species nowadays used at the industrial level for astaxanthin production, or for other microalgal strains engineered to produce ketocarotenoids. Overall, this work identifies a new route to produce astaxanthin on an industrial scale. [ABSTRACT FROM AUTHOR]

Increasing CO2 availability is a common practice at the industrial level to trigger biomass productivity in microalgae cultures. Still, the consequences of high CO2 availability in microalgal cells exposed to relatively high light require further investigation. Here, the photosynthetic, physiologic, and metabolic responses of the green microalga model Chlamydomonas reinhardtii were investigated in high or low CO2 availability conditions: high CO2 enabled higher biomass yields only if sufficient light energy was provided. Moreover, cells grown in high light and high CO2 availability were characterized, compared to cells grown in high light and low CO2, by a relative increase of the energy‐dense triacylglycerols and decreased starch accumulation per dry weight. The photosynthetic machinery adapted to the increased carbon availability, modulating Photosystem II light‐harvesting efficiency and increasing Photosystem I photochemical activity, which shifted from being acceptor side to donor side limited: cells grown at high CO2 availability were characterized by increased photosynthetic linear electron flow and by the onset of a balance between NAD(P)H oxidation and NAD(P)+ reduction. Mitochondrial respiration was also influenced by the conditions herein applied, with reduced respiration through the cytochrome pathway compensated by increased respiration through alternative pathways, demonstrating a different use of the cellular reducing power based on carbon availability. The results suggest that at high CO2 availability and high irradiance, the reducing power generated by the oxidative metabolism of photosynthates is either dissipated through alternative oxidative pathways in the mitochondria or translocated back to the chloroplasts to support carbon assimilation and energy‐rich lipids accumulation. [ABSTRACT FROM AUTHOR]

Light-harvesting complex II (LHCII) serves a central role in light harvesting for oxygenic photosynthesis and is arguably the most important photosynthetic antenna complex. In this work, we present two-dimensional electronic-vibrational (2DEV) spectra of LHCII isolated from spinach, demonstrating the possibility of using this technique to track the transfer of electronic excitation energy between specific pigments within the complex. We assign the spectral bands via comparison with the 2DEV spectra of the isolated chromophores, chlorophyll a and b, and present evidence that excitation energy between the pigments of the complex are observed in these spectra. Finally, we analyze the essential components of the 2DEV spectra using singular value decomposition, which makes it possible to reveal the relaxation pathways within this complex.

We present two-dimensional electronic-vibrational spectra of the chlorophyll (Chl) binding protein light harvesting complex II (LHCII) from spinach, and of isolated Chl a and Chl b. We show how energy transfer from Chl b to Chl a and subsequent relaxation can be directly observed in LHCII using this multidimensional technique.

Light harvesting complex stress-related 3 (LHCSR3) is the protein essential for photoprotective excess energy dissipation (non-photochemical quenching, NPQ) in the model green algaChlamydomonas reinhardtii Activation of NPQ requires low pH in the thylakoid lumen, which is induced in excess light conditions and sensed by lumen-exposed acidic residues. In this work we have used site-specific mutagenesisin vivoandin vitrofor identification of the residues in LHCSR3 that are responsible for sensing lumen pH. Lumen-exposed protonatable residues, aspartate and glutamate, were mutated to asparagine and glutamine, respectively. By expression in a mutant lacking all LHCSR isoforms, residues Asp(117), Glu(221), and Glu(224)were shown to be essential for LHCSR3-dependent NPQ induction inC. reinhardtii Analysis of recombinant proteins carrying the same mutations refoldedin vitrowith pigments showed that the capacity of responding to low pH by decreasing the fluorescence lifetime, present in the wild-type protein, was lost. Consistent with a role in pH sensing, the mutations led to a substantial reduction in binding the NPQ inhibitor dicyclohexylcarbodiimide.

In oxygenic photosynthetic eukaryotes, the hydroxylated carotenoid zeaxanthin is produced from preexisting violaxanthin upon exposure to excess light conditions. Zeaxanthin binding to components of the photosystem II (PSII) antenna system has been investigated thoroughly and shown to help in the dissipation of excess chlorophyll-excited states and scavenging of oxygen radicals. However, the functional consequences of the accumulation of the light-harvesting complex I (LHCI) proteins in the photosystem I (PSI) antenna have remained unclarified so far. In this work we investigated the effect of zeaxanthin binding on photoprotection of PSI-LHCI by comparing preparations isolated from wild-type Arabidopsis thaliana (i.e., with violaxanthin) and those isolated from the A. thaliana nonphotochemical quenching 2 mutant, in which violaxanthin is replaced by zeaxanthin. Time-resolved fluorescence measurements showed that zeaxanthin binding leads to a previously unrecognized quenching effect on PSI-LHCI fluorescence. The efficiency of energy transfer from the LHCI moiety of the complex to the PSI reaction center was down-regulated, and an enhanced PSI resistance to photoinhibition was observed both in vitro and in vivo. Thus, zeaxanthin was shown to be effective in inducing dissipative states in PSI, similar to its well-known effect on PSII. We propose that, upon acclimation to high light, PSI-LHCI changes its light-harvesting efficiency by a zeaxanthin-dependent quenching of the absorbed excitation energy, whereas in PSII the stoichiometry of LHC antenna proteins per reaction center is reduced directly.

In photosynthetic organisms, feedback dissipation of excess absorbed light energy balances harvesting of light with metabolic energy consumption. This mechanism prevents photodamage caused by reactive oxygen species produced by the reaction of chlorophyll (Chl) triplet states with O₂. Plants have been found to perform the heat dissipation in specific proteins, binding Chls and carotenoids (Cars), that belong to the Lhc family, while triggering of the process is performed by the PsbS subunit, needed for lumenal pH detection. PsbS is not found in algae, suggesting important differences in energy-dependent quenching (qE) machinery. Consistent with this suggestion, a different Lhc-like gene product, called LhcSR3 (formerly known as LI818) has been found to be essential for qE in Chlamydomonas reinhardtii. In this work, we report the production of two recombinant LhcSR isoforms from C. reinhardtii and their biochemical and spectroscopic characterization. We found the following: (i) LhcSR isoforms are Chl a/b- and xanthophyll-binding proteins, contrary to higher plant PsbS; (ii) the LhcSR3 isoform, accumulating in high light, is a strong quencher of Chl excited states, exhibiting a very fast fluorescence decay, with lifetimes below 100 ps, capable of dissipating excitation energy from neighbor antenna proteins; (iii) the LhcSR3 isoform is highly active in the transient formation of Car radical cation, a species proposed to act as a quencher in the heat dissipation process. Remarkably, the radical cation signal is detected at wavelengths corresponding to the Car lutein, rather than to zeaxanthin, implying that the latter, predominant in plants, is not essential; (iv) LhcSR3 is responsive to low pH, the trigger of non-photochemical quenching, since it binds the non-photochemical quenching inhibitor dicyclohexylcarbodiimide, and increases its energy dissipation properties upon acidification. This is the first report of an isolated Lhc protein constitutively active in energy dissipation in its purified form, opening the way to detailed molecular analysis. Owing to its protonatable residues and constitutive excitation energy dissipation, this protein appears to merge both pH-sensing and energy-quenching functions, accomplished respectively by PsbS and monomeric Lhcb proteins in plants.

Summary: To investigate the role of intracellular Ca2+ signaling in the perception and response mechanisms to light in unicellular microalgae, the genetically encoded ratiometric Ca2+ indicator Yellow Cameleon (YC3.6) was expressed in the model organism for green algae Chlamydomonas reinhardtii, targeted to cytosol, chloroplast, and mitochondria.Through in vivo single‐cell confocal microscopy imaging, light‐induced Ca2+ signaling was investigated in different conditions and different genotypes, including the photoreceptors mutants phot and acry. A genetically encoded H2O2 sensor was also adopted to investigate the possible role of H2O2 formation in light‐dependent Ca2+ signaling.Light‐dependent Ca2+ response was observed in Chlamydomonas reinhardtii cells only in the chloroplast as an organelle‐autonomous response, influenced by light intensity and photosynthetic electron transport. The absence of blue and red‐light photoreceptor aCRY strongly reduced the light‐dependent chloroplast Ca2+ response, while the absence of the blue photoreceptor PHOT had no significant effects. A correlation between high light‐induced chloroplast H2O2 gradients and Ca2+ transients was drawn, supported by H2O2‐induced chloroplast Ca2+ transients in the dark.In conclusion, different triggers are involved in the light‐induced chloroplast Ca2+ signaling as saturation of the photosynthetic electron transport, H2O2 formation, and aCRY‐dependent light perception. [ABSTRACT FROM AUTHOR]